JNK2 Is a Positive Regulator of the cJun Transcription Factor Anja Jaeschke, Maria Karasarides, Juan-Jose Ventura, Anka Ehrhardt, Chao Zhang, Richard A. Flavell, Kevan M. Shokat, Roger J. Davis  Molecular Cell  Volume 23, Issue 6, Pages 899-911 (September 2006) DOI: 10.1016/j.molcel.2006.07.028 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Comparison of cJun Expression and Phosphorylation in JNK1- and JNK2-Deficient MEFs Wild-type (J1+/+ J2+/+) MEFs and JNK-deficient MEFs (J1−/− J2+/+ and J1+/+ J2−/−) were exposed to UV radiation (50 J/m2). The cells were incubated (30 min), and extracts in TLB buffer were prepared. The expression of JNK, phospho(Thr-183/Tyr-185)-JNK, cJun, and phospho(Ser-63)cJun was examined by immunoblot analysis. JNK protein kinase activity was measured in an in vitro kinase assay with [γ-32P]ATP and cJun as substrates. Molecular Cell 2006 23, 899-911DOI: (10.1016/j.molcel.2006.07.028) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Creation of Mice with a Germ-Line Point Mutation in the Jnk2 Gene (A and B) Strategy for mutating the ATP binding pocket of JNK2. The targeting vector was designed to replace methionine 108 with glycine and to introduce silent mutations that create a SacI restriction site in exon VI. The wild-type allele was replaced with the mutant allele by homologous recombination. Restriction sites are indicated (H, HindIII). The floxed-NeoR cassette used for selection was deleted by using Cre recombinase. (C and D) Genomic DNA isolated from wild-type and targeted ES cells was restricted with HindIII and was examined by Southern blot analysis to demonstrate the correct insertion of the floxed-NeoR cassette into the Jnk2 locus. PCR analysis and restriction with SacI demonstrate the presence of the Jnk2MG allele with a mutation in exon VI. (E) Genomic DNA isolated from wild-type (+/+), heterozygous (+/J2MG), and homozygous (J2MG/MG) knockin mice was examined by PCR. Molecular Cell 2006 23, 899-911DOI: (10.1016/j.molcel.2006.07.028) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Characterization of JNK2MG Inhibition by 1NM-PP1 (A) Primary MEFs prepared from J1−/− J2MG/MG embryos were incubated in medium supplemented with different concentrations of the drug 1NM-PP1 (30 min). The cells were exposed to 50 J/m2 UV radiation and incubated (30 min) with medium supplemented with the drug. The expression of phospho(Ser-63)cJun, cJun, JNK, and tubulin was examined by immunoblot analysis. (B) The inhibition of JNK2MG is reversible. Primary MEFs prepared from J1−/− J2MG/MG embryos were incubated (30 min) with medium supplemented with 10 μM 1NM-PP1. The cells were exposed to 50 J/m2 UV radiation and incubated (30 min) with medium supplemented with the drug. The cells were then washed twice and then incubated in fresh medium without the drug. The cells were harvested immediately (time = 0) or at the indicated time points. The expression of phospho(Ser-63)cJun, cJun, and tubulin was examined by immunoblot analysis. (C and D) 1NM-PP1 is a rapid inhibitor of JNK2MG. Primary MEFs prepared from wild-type embryos (C) or J1−/− J2MG/MG embryos (D) were exposed to 50 J/m2 UV radiation and then incubated in medium for 30 min before the drug 1NM-PP1 (10 μM) or solvent (DMSO) was added to the medium. The cells were harvested immediately after UV treatment (time = −30 min) or at the indicated times (0, 15, and 30 min) after the addition of the drug. The expression of phospho(Ser-63)cJun, cJun, JNK, and tubulin was examined by immunoblot analysis. Molecular Cell 2006 23, 899-911DOI: (10.1016/j.molcel.2006.07.028) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 JNK1 and JNK2 Protein Kinase Activity Contributes to cJun Phosphorylation Primary MEFs prepared from wild-type embryos or mutated embryos (J1+/+ J2MG/MG or J1−/− J2MG/MG) were incubated in medium supplemented with solvent (DMSO) (A) or with 10 μM 1NM-PP1 (B). After 30 min, the cells were exposed to 50 J/m2 UV radiation and then incubated in the same medium for 120 min. The cells were harvested immediately after UV treatment (time = 0 min) or at the indicated times (0, 30, 45, 60, and 120 min) after UV exposure. The expression of JNK, phospho(Thr-183/Tyr-185)-JNK, cJun, phospho(Ser-63)cJun, and tubulin was examined by immunoblot analysis. Molecular Cell 2006 23, 899-911DOI: (10.1016/j.molcel.2006.07.028) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 JNK1 and JNK2 Protein Kinase Activity Contributes to cJun Expression (A) Primary MEFs prepared from wild-type embryos (J1+/+ J2+/+) and mutated embryos (J1+/+ J2MG/MG or J1−/− J2MG/MG) were incubated in medium supplemented with solvent (DMSO) or 10 μM 1NM-PP1 for 48 hr. The expression of cJun, JunB, and JunD mRNA was examined in a ribonuclease protection assay. Control studies were performed to measure the amount of ribosomal protein L32 mRNA. (B) The ribonuclease protection assay data (A) were quantitated by Phosphorimager analysis, and the relative expression of cJun mRNA and JunD mRNA is presented as the normalized ratio [AP1 mRNA]:[L32 mRNA] (mean ± SD; n = 3). (C) Primary MEFs prepared from wild-type embryos (J1+/+ J2+/+) or mutated embryos (J1+/+ J2MG/MG or J1−/− J2MG/MG) were incubated in medium supplemented with solvent (DMSO) or 10 μM 1NM-PP1. The cells were harvested, and the expression of cJun and tubulin was examined by immunoblot analysis. Molecular Cell 2006 23, 899-911DOI: (10.1016/j.molcel.2006.07.028) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 JNK1 and JNK2 Protein Kinase Activities Are Required for Cell Proliferation (A) Primary MEFs prepared from wild-type embryos (J1+/+ J2+/+) or mutated embryos (J1+/+ J2MG/MG or J1−/− J2MG/MG) were incubated in medium supplemented with solvent (DMSO) or 10 μM 1NM-PP1. Relative cell numbers were measured by staining with crystal violet. (B) Primary MEFs were cultured in medium supplemented with solvent (DMSO) or 10 μM 1NM-PP1 for 2 days and then pulse labeled (8 hr) with BrdU. The cells were fixed and stained with an antibody to BrdU (green) and with propidium iodide (red). (C) The expression of cyclin mRNA and ribosomal protein L32 mRNA was examined by ribonuclease protection assay. Molecular Cell 2006 23, 899-911DOI: (10.1016/j.molcel.2006.07.028) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 JNK2 Protein Kinase Activity Promotes Wound Closure In Vitro (A and B) Confluent cultures of primary MEFs prepared from (A) wild-type embryos (J1+/+ J2+/+) or (B) mutated embryos (J1−/− J2MG/MG) were scratched by using a pipette tip to create a wound. The effect of addition of solvent (DMSO) or 10 μM 1NM-PP1 to the culture medium prior to wounding was examined. The wound closure was monitored by video microscopy and representative images taken at 0, 9, and 24 hr are presented (left panels). The phosphorylation of cJun (at 30 min after wounding) at the leading edge of the closing wound was examined by immunofluorescence analysis with an antibody to phospho(Ser-63)cJun (green); nuclei were stained with DAPI (blue) (right panels). (C) Boyden chamber assays were performed by using primary mouse keratinocytes to examine cell migration. The keratinocytes were pretreated (1 hr) and then incubated in Boyden chambers (24 hr) with or without 100 nM 1NM-PP1. The number of cells that migrated to the other side of the membrane was counted. The data represent the mean ± SD of triplicate determinations obtained in one experiment. Similar results were obtained in studies using three independent keratinocytes preparations. (D) Primary MEFs were incubated with medium supplemented with solvent (DMSO) or 10 μM 1NM-PP1 for 48 hr and fixed. Microtubules were stained with an antibody to tubulin (green), filamentous actin was stained with phalloidin (red), and DNA was stained with DAPI (blue). Molecular Cell 2006 23, 899-911DOI: (10.1016/j.molcel.2006.07.028) Copyright © 2006 Elsevier Inc. Terms and Conditions